Development of a sensitive and rapid method for quantitation of (S)-(-)- and (R)-(+)-metoprolol in human plasma by chiral LC–ESI–MS/MS

A selective, sensitive and high throughput liquid chromatography-tandem mass spectrometry(LC–ESI–MS/MS) method has been developed for separation and quantification of metoprolol enantiomers on a chiral Lux Amylose-2(250 mm 4.6 mm, 5 mm) column. Solid phase extraction of(S)-()- and(R)-(t)-metoprolol and rac-metoprolol-d6 as an internal standard(IS)was achieved on Lichrosep DVB HL cartridges employing 200 mL human plasma. Both the analytes were chromatographically separated with a resolution factor of 2.24 using 15 mM ammonium acetate in water, pH 5.0 and 0.1%(v/v) diethyl amine in acetonitrile(50:50, v/v) as the mobile phase within 7.0 min. The precursor-product ion transitions for the enantiomers and IS were monitored in the multiple reaction monitoring and positive ionization mode. The method was validated over the concentration range of 0.500–500 ng/mL for both the enantiomers. Matrix effect was assessed by post-column analyte infusion experiment and the mean extraction recovery was greater than 94.0% for both the enantiomers at all quality control levels. The stability of analytes was evaluated in plasma and whole blood under different storage conditions. The method was successfully applied to a clinical study in 14 healthy volunteers after oral administration of200 mg metoprolol tablet under fasting conditions. The assay reproducibility is shown by reanalysis of 68 incurred samples. The suitability of the developed method was assessed in comparison

Simultaneous quantification of prodrug oseltamivir and its metabolite oseltamivir carboxylate in human plasma by LC-MS/MS to support a bioequivalence study

A simple,precise and rapid liquid chromatography-tandem mass spectrometry(LC-MS/MS) method has been developed and validated for the simultaneous determination of oseltamivir and oseltamivir carboxylate,a neuraminidase inhibitor,using their deuterated analogs as internal standards(ISs).The method involved solid phase extraction of the analytes and ISs from 200 μL human plasma with no reconstitution and drying steps.The chromatographic separation was achieved on a Symmetry C18(100 mm 4.6 mm,5 μm) column using 10 mM ammonium formate and acetonitrile(30:70,v/v) as the mobile phase in a run time of 2.0 min.Quantitation of analytes and ISs were done by multiple reaction monitoring on a triple quadrupole mass spectrometer in the positive ionization mode.The linearity of the method was established in the concentration range of 0.5-200 ng/mL and 2.0-800 ng/mL for oseltamivir and oseltamivir carboxylate respectively.The mean extraction recovery for oseltamivir(94.4%) and oseltamivir carboxylate(92.7%) from spiked plasma samples was consistent and reproducible.The application of this method was demonstratedby a bioequivalence study in 42 healthy Indian subjects with 75 mg oseltamivir phosphate capsules.The assay reproducibility was established by reanalysis of 151 incurred subject samples.

Liquid chromatography-tandem mass spectrometry method for simultaneous determination of albendazole and albendazole sulfoxide in human plasma for bioequivalence studies

An improved high performance liquid chromatography-tandem mass spectrometry(LC-MS/MS) method has been developed for sensitive and rapid determination of albendazole(ABZ) and its active metabolite,albendazole sulfoxide(ABZSO),in the positive ionization mode.The method utilized solid phase extraction(SPE) for sample preparation of the analytes and their deuterated internal standards(ISs) from100 μL human plasma.The chromatography was carried out on Hypurity C_(18) column using acetonitrile-2.0 mM ammonium acetate,pH 5.0(80:20,v/v) as the mobile phase.The assay exhibited a linear response over the concentration range of 0.200-50.0 ng/mL for ABZ and 3.00-600 ng/mL for ABZSO.The recoveries of the analytes and ISs ranged from 86.03%-89.66%and 89.85%-98.94%,respectively.Matrix effect,expressed as IS-normalized matrix factors,ranged from 0.985 to 1.042 for the both analytes.The method was successfully applied for two separate studies in healthy subjects using single dose of 400 mg conventional tablets and 400 mg chewable ABZ tablets,respectively.

LC-UV and LC-MS evaluation of stress degradation behavior of desvenlafaxine

The objective of current study was to develop a validated specific stability indicating reversed-phase liquid chromatographic method for the quantitative determination of desvenlafaxine in bulk sample and pharmaceutical dosage form in the presence of degradation products. Forced degradation studies were performed on bulk sample of desvenlafaxine as per ICH prescribed stress conditions using acid, base, oxidative and photolytic degradation to show the stability indicating power of the method. Significant degradation was observed under acidic stress condition and the degradation product formed was identified by LC-MS and a degradation pathway for drug has been proposed. Successful separation of drug from degradation products formed under stress conditions was achieved on a SymmetryShield column C 18 (5 mm, 250 mm 4.6 mm, i.d.) using the mobile phase consisting of a mixture of 0.2% (v/v) triethylamine in ammonium acetate (0.05 M; pH 6.5) and methanol using isocratic gradient.

Establishment of inherent stability of pramipexole and development of validated stability indicating LC-UV and LC-MS method

Pramipexole belongs to a class of nonergot dopamine agonist recently approved for the treatment of early and advanced Parkinson's disease.A validated specific stability indicating reversed-phase liquid chromatographic method has been developed for the quantitative determination of pramipexole in bulk as well as in pharmaceutical dosage forms in the presence of degradation products.Forced degradation studies were performed by exposition of drug to hydrolytic(acidic and basic),oxidative and photolytic stress conditions,as defined under ICH guideline Q1A(R2).Significant degradation was observed under hydrolytic,oxidative and photolytic conditions and the degradation products formed were identified by LC-MS.

Selective and rapid determination of raltegravir in human plasma by liquid chromatography–tandem mass spectrometry in the negative ionization mode

A selective and rapid high-performance liquid chromatography–tandem mass spectrometry method was developed and validated for the quantification of raltegravir using raltegravir-d3 as an internal standard(IS). The analyte and IS were extracted with methylene chloride and n-hexane solvent mixture from 100 mL human plasma. The chromatographic separation was achieved on a Chromolith RP-18 e endcapped C18(100 mm 4.6 mm) column in a run time of 2.0 min. Quantitation was performed in the negative ionization mode using the transitions of m/z 443.1-316.1 for raltegravir and m/z 446.1-319.0 for IS. The linearity of the method was established in the concentration range of 2.0–6000 ng/m L.The mean extraction recovery for raltegravir and IS was 92.6% and 91.8%, respectively, and the IS-normalized matrix factors for raltegravir ranged from 0.992 to 0.999. The application of this method was demonstrated by a bioequivalence study on 18 healthy subjects.

Simultaneous analysis of allopurinol and oxypurinol using a validated liquid chromatography–tandem mass spectrometry method in human plasma

The present study describes a simple, reliable and reproducible liquid chromatography–tandem mass spectrometry method(LC–MS/MS) for the simultaneous determination of allopurinol and its active metabolite,oxypurinol in human plasma for a pharmacokinetic/bioequivalence study. After protein precipitation(PPT) of100 μL plasma sample with 1.0% formic acid in acetonitrile, the recovery of the analytes and allopurinol-d2 as an internal standard ranged from 85.36% to 91.20%. The analytes were separated on Hypersil Gold(150 mm×4.6 mm, 5 μm) column using 0.1% formic acid-acetonitrile(98:2, v/v) as the mobile phase.Quantification was done using electrospray ionization in the positive mode. The calibration concentration range was established from 60.0 to 6000 ng/m L for allopurinol and 80.0–8000 ng/m L for oxypurinol. Matrix effect in human plasma, expressed as IS-normalized matrix factors ranged from 1.003 to 1.030 for both the analytes. The developed method was found suitable for a clinical study with 300 mg allopurinol tablet formulation in healthy subjects.

An improved LC–MS/MS method for the quantification of alverine and para hydroxy alverine in human plasma for a bioequivalence study

A highly sensitive and selective high performance liquid chromatography–tandem mass spectrometry method was developed and validated for the quantification of alverine(ALV) and its active metabolite, para hydroxy alverine(PHA), in human plasma. For sample preparation, solid phase extraction of analytes was performed on Phenomenex Strata-X cartridges using alverine-d5 as the internal standard. The analytes were separated on Symmetry Shield RP18(150 mm×3.9 mm, 5 μm) column with a mobile phase consisting of acetonitrile and10 mM ammonium formate(65:35, v/v). Detection and quantitation was done by electrospray ionization mass spectrometry in the positive mode using multiple reaction monitoring. The assay method was fully validated over the concentration range of 15.0–15,000 pg/mL for ALV and 30.0–15,000 pg/mL for PHA. The intra-day and inter-day accuracy and precision(% CV) ranged from 94.00% to 96.00% and 0.48% to 4.15% for both the analytes. The mean recovery obtained for ALV and PHA was 80.59% and 81.26%, respectively. Matrix effect,expressed as IS-normalized matrix factor ranged from 0.982 to 1.009 for both the analytes. The application of the method was demonstrated for the specific analysis of ALV and PHA for a bioequivalence study in 52 healthy subjects using 120 mg ALV capsules. The assay reproducibility was also verified by reanalysis of 175 incurred subject samples.

Quantification of 17-desacetyl norgestimate in human plasma by liquid chromatography–tandem mass spectrometry (LC–MS/MS) and its application to bioequivalence study

A rapid and sensitive ultra-performance liquid chromatography–tandem mass spectrometry(UPLC–MS/MS) method was developed and validated for the estimation of 17-desacetyl norgestimate in human plasma using solid-phase extraction technique. 17-desacetyl norgestimate D6 was used as the internal standard. Simple gradient chromatographic conditions and mass spectrometric detection enabled accurate and precise measurement of 17-desacetyl norgestimate at sub-picogram levels. The proposed method was validated for a linear range of 20–5000 pg/m L with a correlation coefficient Z 0.9988. The intra-run and inter-run precision and accuracy were within 10%. The overall recoveries for 17-desacetyl norgestimate and 17-desacetyl norgestimate D6 were 96.30% and 93.90%, respectively. The total run time was 4.5 min. The developed method was applied for the determination of the pharmacokinetic parameters of 17-desacetyl norgestimate following a single oral administration of a norgestimate and ethinyl estradiol0.250 mg/0.035 mg tablets in 35 healthy female volunteers.